Molecular descriptions of mechanosensory mechanisms are limited. The mechanosensitive channel of the large conductance (MscL) of E. coli, the first isolated molecule shown to respond to membrane stretch by opening a large aqueous pore, currently is the most accessible model system. The 0.5 kb gene, the purified protein completely functional when reconstituted in lipid bilayers, a variety of mutants, twenty natural homologs, and, finally, the crystal structure of one homolog are now available. This basic project is aimed at detailed functional characterization of MscL and particularly its homolog from M. tuberculosis (Tb-MscL) recently resolved by X-ray crystallography to 3.5 Angstroms in its closed conformation. It appears that it will be difficult to crystallize the native open channel since the energy of the open state is about 19 kbT above the closed state. The long-term goal of the proposed work is to predict the open conformation and understand the opening process. The specific aims are: (1)To measure the electrophysiological properties of Tb-MscL. (2) Use these results to set constraints for molecular models of the open and subconductance states. The constraints will be obtained from conductances, sieving measurements using polymers, ionic selectivity of the substates and estimates of the changes of in-plane channel expansion associated with the tension dependence of opening. (3) Using effects of co-solvents and site directed mutagenesis to study the nature of intramolecular interactions and role of specific protein motifs that may determine the stretch-sensitivity of MscL. (4) Evaluate energetics of MscL gating in bilayers of different thickness. (5) Evaluate MscL sensitivity to tension in the two monolayers that compose a bilayer since the channel is asymmetric across the bilayer. This will be done by evaluating the gating parameters in conventional 'uncoupled' and 'coupled' bilayers made of archaeal bipolar lipids, and under conditions of a symmetrical bilayer modification. Preliminary computer models of the proteins 3-dimensional structure in open, closed and intermediate conformations have been developed. Testing the critical predictions of these models should clarify the functional role and relationships between different protein domains, and the mechanism by which tension is conveyed from the lipid bilayer to channel gating.